Project Summary/Abstract: Current treatments for ischemic retinopathies are designed to inhibit vascular leakage and proliferation in late disease, but do not target the ischemia and/or inflammation driving pathological neovascularization. A promising alternative approach is the stimulation of functional, physiologic vascular growth and stability. A subset of endothelial progenitor cells called endothelial colony forming cells (ECFCs) can facilitate this. In mouse models of ischemic retinopathy (oxygen-induced retinopathy, OIR), ECFCs provide a significant vasculo- and neurotrophic rescue effect. Several lines of evidence, including published findings from our lab, demonstrate this therapeutic effect to be mediated via paracrine action. However, the details of this mechanism remain uncharacterized. MicroRNA-containing extracellular vesicles (EVs) shed from ECFCs were recently discovered to recapitulate this rescue effect in the OIR model. ECFC-derived EVs promote endothelial cell migration and increase the expression of angiogenesis-related genes. The Friedlander lab has demonstrated that a subset of ECFCs characterized by high expression of the surface marker CD44 (CD44hi) exhibit a more robust vasculo- and neurotrophic rescue effect than ECFCs with low CD44 expression in the OIR model via a paracrine mechanism. In light of these data, the overarching hypothesis of this proposal is that the superior effect of CD44hi ECFCs is attributable, at least in part, to the bioactive microRNA (miR) cargo in EVs released from this ECFC subpopulation. I propose to investigate which miRs are upregulated in EVs shed from CD44hi ECFCs in order to identify novel miRs responsible for the reported neurovasculotrophic effects of ECFC EVs. EVs will then be engineered to increase expression of these candidate miRs with the goal of re-designing therapies that achieve even greater therapeutic effects in the OIR model. As natural carriers of miRs, EVs may be a highly effective drug delivery vehicle that circumvents the risks of tumorigenesis associated with cell-based therapeutics. ECFC-derived EVs home to sites of ischemia/angiogenesis, potentially limiting any deleterious side effects of systemic delivery. However, a greater understanding of heterogeneous populations of EV and the relationship between their cargo and functional effects must be developed before translation into the clinic can become a reality.